We previously designed a modified channelrhodopsin-1 (mVChR1) protein chimera with a broader action than that of Chlamydomonas channelrhodopsin-2 and reported that its transduction into retinal ganglion cells can restore visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats, with photostimuli ranging from 486 to 640 nm. In the current study, we sought to investigate the safety and influence of mVChR1 transgene expression. Adeno-associated virus type 2 encoding mVChR1 was administered by intravitreous injection into dystrophic RCS rats. Reverse-transcription PCR was used to monitor virus and transgene dissemination and the results demonstrated that their expression was restricted specifically within the eye tissues, and not in non-target organs. Moreover, examination of the blood, plasma and serum revealed that no excess immunoreactivity was present, as determined using standard clinical hematological parameters. Serum antibodies targeting the recombinant adeno-associated virus (rAAV) capsid increased after the injection; however, no increase in mVChR1 antibody was detected during the observation period. In addition, retinal histological examination showed no signs of inflammation in rAAV-injected rats. In conclusion, our results demonstrate that mVChR1 can be exogenously expressed without harmful immunological reactions in vivo. These findings will aid in studies of AAV gene transfer to restore vision in late-stage retinitis pigmentosa.

Previous studies have indicated that Müller glia in chick and fish retinas can re-enter the cell cycle, express progenitor genes, and regenerate neurons via the Notch signaling pathway in response to retinal damage or growth factors. Here, we investigated the role of Notch signaling and the effect of hypoxia, as a means to induce retinal damage, on the proliferation of an immortalized Müller cell line (rMC-1 cells). Our data showed that rMC-1 cells expressed Müller glia and neural and retinal progenitor markers but did not express neuronal or retinal markers. Hypoxia increased rMC-1 cell proliferation by activating the positive cell-cycle regulators, cyclins A and D1, as well as the neural and retinal progenitor markers, Notch1, Hes1, nestin, Sox2, Msi1, Pax6, and NeuroD1. However, hypoxia did not significantly influence the expression of Müller glial markers GS, CRALBP, and cyclin D3 or the death of the rMC-1 cells. The increase in cell proliferation induced by hypoxia was greatly attenuated by blocking Notch signaling with the inhibitor DAPT, resulting in the reduced expression of positive cell-cycle regulators (cyclins A and D1) and neural and retinal progenitor markers (Notch1, Hes1, Sox2, Pax6, and NeuroD1). Blockade of the Notch signaling pathway by DAPT after hypoxia promoted the differentiation of rMC-1 cells to neurons, as demonstrated by the induction of neural marker (Tuj1), retinal amacrine (Syntaxin1), and retinal ganglion cell (Brn3b) markers, although the expression of the latter marker was low. Taken together, our data indicate that Notch signaling is required for proliferation under hypoxic conditions either by activating the positive cell-cycle regulators or by skewing their de-differentiation towards a neural progenitor lineage. These findings indicate that the Notch signaling pathway regulates hypoxia-induced proliferation and differentiation of Müller glia.

We had previously reported that transduction of the channelrhodopsin-2 (ChR2) gene into retinal ganglion cells restores visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats. In this study, we attempted to reveal the safety and influence of exogenous ChR2 gene expression. Adeno-associated virus (AAV) type 2 encoding ChR2 fused to Venus (rAAV-ChR2V) was administered by intra-vitreous injection to dystrophic RCS rats. However, rAAV-ChR2 gene expression was detected in non-target organs (intestine, lung and heart) in some cases. ChR2 function, monitored by recording visually evoked potentials, was stable across the observation period (64 weeks). No change in retinal histology and no inflammatory marker of leucocyte adhesion in the retinal vasculature were observed. Although antibodies to rAAV (0.01–12.21 μg ml−1) and ChR2 (0–4.77 μg ml−1) were detected, their levels were too low for rejection. T-lymphocyte analysis revealed recognition by T cells and a transient inflammation-like immune reaction only until 1 month after the rAAV-ChR2V injection. In conclusion, ChR2, which originates from Chlamydomonas reinhardtii, can be expressed without immunologically harmful reactions in vivo. These findings will help studies of ChR2 gene transfer to restore vision in progressed retinitis pigmentosa.

BACKGROUND:We have demonstrated a culture bag system that is useful for pancreatic islet transplantation. To improve and simplify islet transplantation procedures from culture to transplantation, we developed a novel device specific for both islet culture and transplantation (TUBERO Device [TD]) using an oxygen-permeable material.MATERIALS AND METHODS:Porcine islets with 30 minutes warm ischemia time were cultured for 24 hours at 37 degrees C in 5% CO2 and humidified air under three different procedures: (1) ordinary culture flask, (2) culture bag suitable for platelets, and (3) TD. Loss of islets during culture, glucose-stimulated insulin release as an islet functional test, and ADP/ATP ratio as an index of islet viability tests were evaluated to compare the devices. TD was further applied in two clinical islet transplantations using non-heart-beating donors in Japan.RESULTS:The loss of islets during culture was considerably lower in the TD group. The stimulation index upon glucose challenge tests was significantly higher in the TD group than the others. The ADP/ATP ratio in TD group was significantly lower than that in the ordinary flask group, suggesting that the apoptotic islets were relatively lower among TD. Most importantly, TD was successfully applied both in the clinical islet cultures and in transplantation, resulting in excellent graft function.CONCLUSIONS:We propose that the TD, a novel product, not only simplifies islet transplantation procedures, but also maintain the quality of isolated islets.

To recover visual sensation of blind patients, we have fabricated a fully implantable retinal prosthesis chip that includes photodetector and stimulus current generator. For the first time, we successfully implanted the retinal prosthesis chip bonded on the flexible cable with stimulus electrode array into a rabbit eyeball. Moreover, we recorded and analyzed electrically evoked potential (EEP) elicited from a rabbit brain by current stimulation to retina.

Arrestin is a 45 KDa protein and is mainly located in the membrane of photo-receptor rod cells and pineal gland. It is well known that 1147delA nutation in the arrestin gene causes Oguchi disease and autosomal recessive retinitis pigmentosa in Japanese patients. We examined expression of arrestin in mRNA isolated from whole blood cells of patients with Oguchi disease, who had the 1147delA mutation in the arrestin gene. Arrestin was expressed at the level of mRNA in all of three patients with Oguchi disease. mRNA from Oguchi patients did not have the same 1147delA mutation as was seen in the arrestin gene but had the normal sequence. The result indicated the possibility of RNA editing in blood cells in our patients.

PURPOSE The aim of this study was to determine whether indocyanine green (ICG) is toxic to cultured human retinal pigment epithelial (ARPE-19) cells, and whether hypothermia can protect the ARPE-19 cells against the ICG toxicity. METHODS Cultured ARPE-19 cells were exposed to 0.25, 0.5, 1, 2.5, and 5 mg/mL of ICG dye at 37 and 4 degrees C for 30 min. The percentage of ARPE-19 cells that survived was determined by resazurin 1 day after the exposure. RESULTS Exposure of the RPE cells to a hypotonic saline solution with an osmolarity equal to 5 mg/mL of ICG did not induce a statistically significant decrease in the percentage of RPE cells that survived. Exposure of the ARPE-19 cells to ICG induced a significant decrease in the percentage of cell survival at all concentrations of ICG (P<0.05), except in 0.25 mg/mL at 37 degrees C. At 4 degrees C, on the other hand, ICG induced a statistically significant decrease in the percentage of RPE cell survival only at 5 mg/mL of ICG (P<0.05). CONCLUSIONS These results indicate that ICG is toxic to human RPE cells in culture, and that cell death cannot be attributed to the low osmolarity. Hypothermia of 4 degrees C has a protective effect against ICG toxicity.

PURPOSE:To determine whether hypothermia of 8 degrees C can protect cultured human retinal pigment epithelial (ARPE-19) cells and rat retinal ganglion cells (RGC-5) against trypan blue (TB) toxicity.DESIGN:Laboratory investigation.METHODS:ARPE-19 cells and RGC-5 were exposed to balanced salt solution as controls, and 0.05% and 0.5% TB at 37 degrees C, and at 8 degrees C for one minute. The percentage of surviving cells was determined by the resazurin test.RESULTS:TB induced a statistically significant decrease in the percentage of ARPE-19 cells surviving at 0.5% TB at 37 degrees C (P < .01). Conversely, TB induced a statistically significant decrease in the percentage of RGC-5 surviving at all conditions except for 0.05% TB at 8 degrees C (0.05% 37 degrees C; P < .05, 0.5% 37 degrees C and 8 degrees C; P < .01).CONCLUSIONS:These results indicate that reducing the temperature to 8 degrees C has a protective effect against the TB toxicity for ARPE-19 cells and RGC-5 in culture.

We have proposed a novel retinal prosthesis system with three-dimensionally stacked retinal prosthesis chip. The retinal prosthesis chip consists of several LSI chips that are vertically stacked and electrically connected using three-dimensional integration technology. We fabricated retinal prosthesis chip including photodetectors and stimulus current generators. We confirmed that current waveform parameters can be varied by bias voltages. Implantable stimulus electrode array was also fabricated for the electrical stimulation of the retina. To evaluate optimal retinal stimulus current, electrically evoked potential (EEP) was recorded in animal experiments. The recorded waveform shows a similar behavior to the visually evoked potential (VEP) waveform, indicating possibilities that the electrical stimulation of the retina can restore visual sensation for the blind patients

PURPOSE To determine whether hypothermia of 8 degrees C can protect cultured human retinal pigment epithelial (ARPE-19) cells and rat retinal ganglion cells (RGC-5) against trypan blue (TB) toxicity. DESIGN Laboratory investigation. METHODS ARPE-19 cells and RGC-5 were exposed to balanced salt solution as controls, and 0.05% and 0.5% TB at 37 degrees C, and at 8 degrees C for one minute. The percentage of surviving cells was determined by the resazurin test. RESULTS TB induced a statistically significant decrease in the percentage of ARPE-19 cells surviving at 0.5% TB at 37 degrees C (P < .01). Conversely, TB induced a statistically significant decrease in the percentage of RGC-5 surviving at all conditions except for 0.05% TB at 8 degrees C (0.05% 37 degrees C; P < .05, 0.5% 37 degrees C and 8 degrees C; P < .01). CONCLUSIONS These results indicate that reducing the temperature to 8 degrees C has a protective effect against the TB toxicity for ARPE-19 cells and RGC-5 in culture.

Royal College of Surgeons (RCS) rats have been extensively used as an animal model of a recessively inherited retinal degeneration. RCS rats have a genetic defect that leads to phagocytosis of photoreceptor outer segments by the retinal pigment epithelial cells that then results in retinal degeneration. In 2000, positional cloning showed that RCS rats had a deletion of the MERTK gene, which encodes a receptor tyrosine kinase [1]. Subsequently, patients with retinal dystrophies were screened for mutations in the human ortholog of the MERTK gene, and three disease-causing mutations and 11 sequence variants were detected [2]. To date, there have been only three published investigations of mutational screening of the MERTK gene on patients with inherited retinal degeneration [2-4]. Interestingly, mutations in the MERTK gene have not been reported in Japanese patients with autosomal recessive retinitis pigmentosa (arRP). The purpose of this study was to determine whether mutations in the MERTK gene were present in Japanese patients with arRP. To accomplish this, we screened 96 patients who were diagnosed to have arRP clinical findings.

PURPOSE: To describe the clinical and genetic characteristics of six Japanese families with Bietti's crystalline corneoretinal dystrophy (BCD).DESIGN: Case reports and results of DNA analysis.METHODS: Mutation screening was performed on six unrelated patients with BCD by direct sequencing. The clinical features were characterized by the visual acuity, slit,lamp biomicroscopy, electroretinography, fluorescein angiography, and kinetic visual field testing.RESULTS: An identical IVS6 to 8deITCATACAGGTCATCGCG/insGC mutation in the CYP4V2 gene was identified in five of the patients with BCD; the sixth patient had a novel Trp340X mutation in the CYP4V2 gene. Three patients showed crystalline-like deposits at the limbus by specular microscopy. Ophthalmic findings of all patients had a rapid progression after age 50 years.CONCLUSIONS: Our findings suggest that the IVS6 to 8delTCATACAGGTCATCGCG/insGC mutation is a common mutation in Japanese patients with BCD. Although phenotypic variability was found, the natural course was almost the same in all of our patients. (c) 2005 by Elsevier Inc. All rights reserved.

Methods. Rat eyes were enucleated on embryonic day 18 and on postnatal days 1, 4, 9, 15, 19, and 90. The avidin-biotin-peroxidase technique was applied to show aB-crystallin immunostaining. After the cornea and lens were removed, the developing rat eyeballs were solubilized by 1% sodium dodecyl sulfate. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) were performed to detect aB-crystallin in the extracts.

We report a patient with a spontaneous intramuscular hematoma in the lateral rectus muscle of the eye that resolved without medication with maintenance of good vision. A 40-year-old woman presented with ocular pain and exophthalmos in her right eye. She had no history of trauma or surgery. Exophthalmos and limitation of abduction and supraduction of her right eye were present at the initial examination. Magnetic resonance (MR) images showed an intramuscular hematoma in the right lateral rectus muscle. Her other ocular findings were within normal limits. Five months later without any treatments, the MR images were within normal limits, and her ocular signs and symptoms were completely resolved. Careful observations including MR imaging is sufficient for patients with a spontaneous intramuscular hemorrhage in the extraocular muscle, and the visual prognosis is good.

PURPOSE. To establish an efficient method of transferring the human brain-derived neurotrophic-factor (hBDNF) gene into human iris pigment epithelial (hIPE) cells by using recombinant adeno-associated virus type 2 (rAAV2).METHODS. Cultured hIPE cells were treated with either hydroxyurea-sodium butyrate (HUSB; DNA synthesis inhibitor), or tyrphostin-1 (Tyr; epidermal growth factor receptor [ EGFR] tyrosine kinase inhibitor), or a combination of HUSB and Tyr (HUSB-Tyr). After each treatment, cells were exposed to rAAV2 (rAAV-LacZ or rAAV-hBDNF). The levels of BDNF were measured by ELISA and also determined by Western blot analysis. Southern blot analysis was performed on each type of treated cell. The neuroprotective effect of BDNF on the retinal ganglion cells (RGCs) was quantitatively assessed by culturing rAAV-hBDNF-hIPE with RGCs.RESULTS. The infection of hIPE cells was significantly lower than ARPE and HT1080 cells, which are highly permissive cells for rAAV2. The treatment of HUSB-Tyr enhanced the transgene expression more than that after treatment with one of these agents in rAAV-hIPE cells. Southern hybridization revealed that the amount of replicative form monomer (RFm) was less in Tyr than in HUSB or HUSB-Tyr treatment and there was no difference in conversion of virus genome to double stranded form after HUSB and HUSB-Tyr treatment. However, adding Tyr treatment stimulated the JNK1/2 and p38 pathways and modified the target transgene expression. BDNF had a significantly greater rescue effect of RGCs with the HUSB-Tyr-treated rAAV-hBDNF-hIPE cells ( P < 0.01) than that with the HUSB-treated rAAV-hBDNF-hIPE cells ( P > 0.05) compared with noninfected hIPE cells.CONCLUSIONS. The combined treatment of HUSB-Try is an effective method of increasing transgene expression with the AAV-mediated gene transfer. The role of HUSB and Tyr in the increase of gene expression may be different and related to the conversion of virus into the host genome and the enhancement of the transcription, respectively.